Technologies for conjugating PEG to peptides and proteins are based on Davis and Abuchowski study (Abuchowski A. et al., J. Biol. Chem., 252, 3571-3581, 1977; Abuchowski A. et al., J. Biol. Chem., 252, 3582-3586, 1977). PEG is a polymer which is hydrophilic, biocompatible and harmless having the structure of H(OCH2CH2)nOH and it is known that in case of being conjugated to peptide and protein, it inhibits enzymatic metabolism via steric hindrance as the same way as sugar chain in glycoprotein does, decreases renal glomerular filtration with increased molecular size of peptide and protein having conjugated PEGs, thereby increasing the duration of physiological activity. Covalent bond between polypeptide and PEG was published in U.S. Pat. No. 4,179,337, and it was described that modification of proteins and enzymes with PEG leads to reduced immunogenicity and antigenicity and increased half life within blood.
For covalent bonding of PEG to polypeptide, it is necessary that “activation” process of converting the terminal hydroxyl moiety of PEG to a reactive functional group. As an “activated PEG”, alkylating agents such as PEG aldehydes, PEG epoxides and PEG tresylates, and acylating agents such as PEG esters can be enumerated, and the representative example is PEG succinimidyl succinate. Poly(ethyleneglycol)-N-succinimide carbonate and producing method thereof was known from Abuchowski et al., Cancer Biochem. Biophys. 7:175-186(1984) and U.S. Pat. No. 512,614 etc. PEGs in molecular range of 2,000-40,000 can be employed and PEGs such as methoxylated PEGs and branched PEGs can be used. The branched PEG can be represented with the structure of R(-PEG-OH)m [herein, R defines central core moiety such as pentaerythritol or glycerol, m defines the number of branching arm in a range of 3-100]. Hydroxyl group can be used for chemical modification. Other branched PEG with the structure of (CH3O-PEG-)pR—X (WO96/21469) is employed [herein, p is 2 to 3, R represents central core moiety such as lysine and glycerol, and X defines a functional group such as carboxyl group used for activation. Pendant PEG of another branched PEG has functional group such as carboxyl moiety on PEG backbone, other than terminus of PEG chain. All these branched PEGs can be used via “activation” as described previously.
In the reaction of conjugating “activated” PEGs to peptide amines, generally, PEGs bond to one or more amines nonspecifically, thus the core of this technology lies in the method of specifically combining PEG molecules to amines at specific positions. However, in case of peptides containing at least one lysine within the amino acid sequence, as two or more amines exist therein, it is very difficult to specifically conjugate PEGs to amines at specific sites. In case PEGs are conjugated to peptide according to general methods, various conjugates can be generated, from a conjugate having one conjugated PEG to a conjugate having multiple-conjugated PEGs as many as the number of amines, and even in case of the conjugate having one PEG (a mono-PEG conjugate), positional isomers in which conjugation sites of PEG are different one another, can be formed. Usually, as PEG conjugates of peptide differ in activity and enzymatic metabolism according to the number of combined PEGs and combined sites thereof, the method producing such mixture containing various conjugates is disadvantageous for the reason of very low activity yield and being a mixture. To separate and purify specific isomer from the mixture is very complicated, accompanied by very low yield and high cost. To compensate such disadvantages, various methods for site-specific PEGylation have been proposed, however, efficient methods for specific conjugation of PEGs to amines on specific positions has not yet been developed.
Chem. Pharm. Bull. 39(12): 3373-3375(1991) reported on a conjugate of fibronectin-related tripeptide(Arg-Gly-Asp) and amino-poly(ethyleneglycol) and activity thereof. Herein, a method for preparing PEG conjugate by combining amino PEG to aspartic acid activated with dicyclohexylcarbodiimide(DCC)/1-hydroxybenzotriazole(HOBt), was proposed. However, this method is defective in that it can be used only for modification of C-terminus and racemization of adjacent amino acids can occur during the C-terminal activation of peptide. Such racemization can occur in every amino acids except glycine, resulting in reduction of activity of peptide.
Methods for combining monomethoxy poly(ethyleneglycol)(mPEG) or polyvinylalcohol(PVA) to peptide using synthetic resin were published in Journal of Protein Chemistry 10(6):623-627(1991). Since only the conjugate in which a single polymer is combined to N-terminus of the peptide from the method, it fails to provide a method for combining PEG to positions that can maximize the effect of PEGylation (maximizing the duration of peptide activity and minimizing enzymatic metabolism).
PCT patent application No. PCT/US94/06953(1994) provides methods for preparing site-specific PEG peptide in which PEG is introduced during synthetic process of the peptide. That is, it provides a method for preparing PEG-conjugated peptide in which peptide is partially synthesized until its sequence reaches lysine at targeted position, and then PEG is combined, and finally the remaining portion of the peptide is synthesized to complete PEG peptide, a method for preparing PEG-conjugated peptide by using, as the lysine of targeted site, lysine in which Nα-amine thereof was protected with Fmoc(9-fluorenylmethoxycarbonyl) and branched amine thereof was combined with PEG, and a method wherein peptide fragment having PEGs combined to the amines of targeted sitse and the remaining peptide fragment were separately synthesized and combined to complete the PEG-conjugated peptide. Nevertheless, such methods might bring undesirable result due to the effect of the previously combined PEG on subsequent step of the synthesis. For example, in case PEG is introduced during the peptide synthesis and synthesis of the remaining portion is to be continued, a peptide lacking at least one amino acid of the amino acid sequence might be produced, and such peptide can b harmful to a living body and its purification is almost impossible. Therefore, such method is not desirable as a method for preparing a PEG-conjugated peptide usable as a medicine.
PCT patent application No. PCT/EP98/07748 provides methods for combining PEG to amine of GRF in an organic solvent and purifying the resulting mixture of Lys(PEG)12-GRF, Lys(PEG)21-GRF, Lys(PEG)12,21-GRF and [Nα-PEG-Try1, Lys(PEG)12,21]-GRF by gel chromatography and reverse phase chromatography. However, this method cannot provide specificity between amine moieties of Nα, Lys12 and Lys21. Said patent also provides methods for synthesis of [Lys(Alloc)12,21]-GRF(1-29) and [Nα-isopropyl-Try1, Lys(Alloc)12]-GRF(1-29) and specific PEGylation using them, though, in case of [Lys(Alloc)12,21]-GRF(1-29), PEG can be conjugated to only Nα amine, and fails to provide a preparing method for Lys21-PEG conjugate which is most effective as described in said patent. In addition, in case PEG conjugate is prepared by using [Nα-isopropyl-Try1, Lys(Alloc)12]-GRF(1-29), not PEG conjugate of GRF(1-29)-NH2 but PEG conjugate of [Nα-isopropyl-Try1]-GRF(1-29)-NH2, i.e. [Nα-isopropyl-Try1, Lys(PEG)21]-GRF(1-29) is to be formed. Therefore, this method fails to provide completely specific PEGylation for the original peptide having N-terminal amine, GRF(1-29)-NH2.
The inventors of the present invention intended to develop specific PEGylation by which product yield can be noticeably raised and efforts and cost needed for separation and purification of the final product can be reduced, by forming the final product in which PEGs are conjugated only to amines at targeted sites while preventing the formation of impurity product having PEGs conjugated to amines of untargeted sites.
The present invention provides method for preparing specific PEG-conjugated peptide in which the effect of PEG conjugation is maximized by allowing PEGs to be conjugated to amines at targeted sites only with complete selectivity.